DSpace Collection:http://hdl.handle.net/2381/387
Thu, 08 Feb 2018 04:34:32 GMT2018-02-08T04:34:32ZPredicting radiotherapy toxicity in patients treated with radical radiotherapy using predictive assays and circadian rhythmhttp://hdl.handle.net/2381/40982
Title: Predicting radiotherapy toxicity in patients treated with radical radiotherapy using predictive assays and circadian rhythm
Authors: Johnson, Kerstie Anne
Abstract: Radiotherapy is a fundamental cancer treatment and plays a pivotal role in the improving outcomes for the disease. Depending on the cancer site and organ at risk rates of moderate to severe acute toxicity range between 15 to 30 percent and rates of late toxicity between 5 and 15 percent. If a patient’s individual risk of radiotherapy toxicity could be predicted then their treatment could be tailored appropriately.
In this thesis two cohorts have been used to analyse predictive measures for acute and late radiotherapy toxicity: the REQUITE cohort (prospective international observational study of breast, prostate and lung cancer patients) and the LeND cohort (retrospective local study of breast cancer patients). Three main areas have been examined to establish whether they can be used to predict for radiotherapy reactions. In the prostate and lung patients associations between clinical and treatment variables and acute toxicity were reviewed. The second area was predictive assays: DNA damage and repair were assessed using the comet and γ-H2AX assays and apoptosis in lymphocytes using the RILA (radiation induced lymphocyte apoptosis assay). Finally the effect of circadian rhythm and its underlying genetics on radiotherapy toxicity were assessed.
Many of the variables were significantly associated with increased toxicity on univariate analysis. Three were significantly associated with toxicity on multivariate analysis. Acute toxicity in prostate patients was associated with intended duration of hormones (p=0.05) and V50 bladder (p=0.01)). Morning radiotherapy was associated with increased overall bivariate STAT score (p=0.03) in the LeND volunteers.
The results of this study indicate clinical and genetic variables and the use of predictive assays can be utilised to create more personalised radiotherapy treatments that strive for better cancer and quality of life outcomes for patients.Tue, 23 Jan 2018 15:40:25 GMThttp://hdl.handle.net/2381/409822018-01-23T15:40:25ZEvolution of Plant Male Germline-Specific Transcription Factor DUO POLLEN 1http://hdl.handle.net/2381/40861
Title: Evolution of Plant Male Germline-Specific Transcription Factor DUO POLLEN 1
Authors: Zhao, Mingmin
Abstract: Flowering plants account for the 30 crops that provide 95 % of the food for humans. The reproduction of this group depends on the production of two twin sperms. The establishment of the male germline lineage requires the transcription factor DUO POLLEN 1 (DUO1). DUO1 is required for both the cell cycle progression and sperm cell differentiation. This thesis focused on the origin of DUO1 and its target regulation.
Much work was dedicated in searching the evolutionary origin of DUO1 in the R2R3 MYB clade. Based on the analysis of sequences homologous to DUO1 and its sister clade GAMYB, the earliest DUO1 homolog was identified in the green algae. The DUO1 clade did not proliferate after multiple polyploidy events, possibly restricted by its male germline-specific role supported by transcriptome data. The ancestral DUO1 experienced a major MYB domain sequence change in the bryophytes and a second change in the C-terminus in the angiosperms. The MYB domain changes caused a change in the target DNA sequence, which has then been conserved among Embryophyta DUO1 homologs. Another change also happened in the region where a miR159 binding site is present in most angiosperm DUO1 homologs. Sequence and functional analysis showed that this change evolved long before the emergence of miR159. The changes in the C-terminus of DUO1 led to a higher target promoter activation capability in the angiosperm homologs, which was confirmed by functional tests of the angiosperm and bryophyte DUO1. This C-terminal region contains the transactivation domain (TAD) of DUO1 and certain functionally important motifs were highlighted in the study. While these motifs indicated that DUO1 was a member of a TAD family, it was also demonstrated that unknown sequences carry critical features for activation. Together these results mapped the evolution history of DUO1 in the Streptophyta lineage.Mon, 15 Jan 2018 12:36:52 GMThttp://hdl.handle.net/2381/408612018-01-15T12:36:52ZCopy Number Variation and Relevance to Disease of the Complement C3b/C4b Receptor 1 (CR1) Genehttp://hdl.handle.net/2381/40701
Title: Copy Number Variation and Relevance to Disease of the Complement C3b/C4b Receptor 1 (CR1) Gene
Authors: Kucukkilic, Ezgi
Abstract: The complement 3b/4b receptor 1 (CR1) gene is located at chromosome 1q32.2 in a cluster of complement-related genes. CR1 regulates both classical and alternative pathways of the complement system. CR1 is a major receptor for Plasmodium falciparum, and variation within the gene has been associated with different malarial clinical phenotypes. CR1 shows intragenic copy number variation (CNV) resulting in variation in protein length and number of C3b/C4b binding domains. Previously, CR1 was related to Alzheimer’s disease (AD) via complement system regulation. Furthermore, CR1 variation is responsible for the alleles of the Knops blood group, including McCoy and Swain-Langley.
In this thesis, Novel paralogue ratio test (PRT) assays were developed to robustly type CNV of the low-copy repeat (LCR) regions (which defines the common CR1-A and CR1-B alleles, but also rarer alleles) within the gene in large cohorts, and an allele-specific hybridisation assay to genotype alleles of the Knops blood group system.
Variation was analysed across global populations, and in the Tori-Bossito cohort (563 infants) from Benin, followed since birth to observe malaria acquisition and treatment. This showed that the Swain-Langley Sl2 polymorphism is not in strong linkage disequilibrium (LD) with the CNV, nor with other Knops blood group alleles. It appears to provide protection against early acquisition of malaria and subsequent number of malarial infections in the Tori-Bossito cohort but these results were not confirmed in an independent cohort (n=276).
The association between the CR1-B allele and AD (early-onset (EOAD) and late-onset (LOAD)) was explored, showing that the CR1 risk loci (rs3818361, rs6656401 (only for EOAD) and rs6701713) were in moderate LD with CR1-B, but revealing no association between CR1-B (p=0.755) and EOAD (n=633). However, the CR1-B allele (risk) appears to be associated with LOAD (n=2185) with (p=0.015) and without (p=0.048) use of a junction fragment PCR assay.Tue, 02 Jan 2018 16:11:13 GMThttp://hdl.handle.net/2381/407012018-01-02T16:11:13ZBioinformatics analyses of genetic variation in genomes of Neisseria meningitidis (the meningococcus)http://hdl.handle.net/2381/40692
Title: Bioinformatics analyses of genetic variation in genomes of Neisseria meningitidis (the meningococcus)
Authors: Al-Maeni, Mohammad Abdul Rahmman Mohammad
Abstract: Genetic variation is one of the key concepts underlying persistence of Neisseria meningitidis in its host and counteracting both innate and adaptive immune responses of the host. The mechanism of evolution involves the combined action of de novo mutation, recombination, and localised hypermutation. This study aimed to understand the contributions of these processes to within host evolution during host persistence of N. meningitidis for a period of months. A study of 40 isolates from one carrier and representing six months persistent carriage showed that de novo mutation resulting in single nucleotide polymorphisms (SNPs) was the major factor in structuring of the population. Allelic variants were subject to dynamic temporal fluctuations through persistence of meningococcal isolates over several months. Conversely, recombination was found to a powerful mechanism for generating SNPs and insertion/deletion within 25 paired isolates from 25 carriers and representing between 1 and 6 months host persistence. The processes of de novo mutation and recombination were infrequent but exhibited trends toward surface antigens especially pilin, porins, iron acquisition and capsule genes. Variation in intergenic regions was also examined in these isolates and a high level of variation was observed in conserved functional patterns of Corriea elements. Three carriers were examined for changes in expression of three phase variable genes (opc, hpuAb, and nalP); these were in the OFF state indicating that there may have been selection for low expression. A further investigation of microevolution within a clonal complex found a low rate of recombination within the 25 CC-174 disease and carriage isolates but it was many folds higher than recombination rates of species forming clonal population. Variable genes were distributed into several schemes including bacterial secretion systems, iron acquisition, capsule, surface antigen, bacterial mobility proteins, and antimicrobial resistance, and toxin genes. In conclusion, all the processes of evolution de novo mutation, recombination, and localized hypermutation facilitate asymptomatic carriage of meningococci and microevolution of CC-174.Wed, 20 Dec 2017 13:04:58 GMThttp://hdl.handle.net/2381/406922017-12-20T13:04:58ZInvestigating reproductive development in Brachypodium distachyon focussing on the YABBY family of transcription factorshttp://hdl.handle.net/2381/40354
Title: Investigating reproductive development in Brachypodium distachyon focussing on the YABBY family of transcription factors
Authors: Yusoff, Syabira
Abstract: Brachypodium, as a sister to the core pooids containing wheat, barley, oats and rye, represents a good model and point of comparison for the study of development and evolution in temperate cereals. Using a comparative cellular developmental and transcriptomic approach, we investigated regulation of key stages in grain development. This was achieved by generating a transcriptome incorporating several distinct developmental stages of Brachypodium grains; pre-anthesis ovaries, young grain (1-3 DAA), mid-length grain (3-8 DAA), full-length (8-15 DAA) and mature grain (15-24 DAA), mature grain (without embryo), germinating grains and seedlings stage. By looking at the differential expression of genes through grain development we identified clusters that coincide with the initiation of key developmental stages, such as the initiation of endosperm proliferation, cellularisation and differentiation, as well as the activation of specific metabolic pathways, such as starch and protein biosynthesis. Focus was given to members of the YABBY gene family that have an established role in promoting abaxial cell fate and as master regulators of reproductive development in eudicots, but with less clarity in grass species. Using Brachypodium as the model plant for cereal crops, the orthologues of YABBY genes in grasses were identified and subjected to detailed phylogenetic, expression and functional analyses using Bayesian Interference (BI) analyses, RT-PCR, transcriptomics, mRNA in situ hybridization (ISH) and RNAi. Based on several analyses, YABBY6 was suggested as a novel candidate of transcription factors regulating seed development in Brachypodium. Metadata from Chapter 2 were used to extract similar expression genes of YABBY family members and potential motifs regulated in polarity networks involving YABBY genes were suggested.Mon, 11 Sep 2017 14:28:44 GMThttp://hdl.handle.net/2381/403542017-09-11T14:28:44ZThe Type I Restriction Modification System SpnIII of Streptococcus pneumoniaehttp://hdl.handle.net/2381/40353
Title: The Type I Restriction Modification System SpnIII of Streptococcus pneumoniae
Authors: De Ste Croix, Megan
Abstract: The phase variable expression of alternative specificity subunits by the type I restriction modification system SpnIII has been proven to differentially regulate important virulence factors. These gene expression differences are envisaged to translate into significant differences in the virulence of strains, dominated by different specificity subunits, in murine models of infection. The presence of this type I system in the core pneumococcal genome hints at a conserved, global mechanism of regulation, including that of virulence.
The use of wildtype strains enriched in alternative specificity subunits has revealed that recombination within the spnIII locus is not reciprocal and is highly variable between strains. Despite this variability, methylation of the genome by different SpnIII variants results in differential gene expression and significant differences in models of invasive disease. The recombination within the spnIII locus appears to be much more complex than similar, previously reported systems. Recombination is not mediated by the classical homologous recombination pathways but is partially controlled by a site specific tyrosine recombinase.
Investigations into the impact of SpnIII may be capable of improving our understanding of how this nasopharyngeal coloniser is capable of causing devastating invasive disease.Mon, 11 Sep 2017 14:22:07 GMThttp://hdl.handle.net/2381/403532017-09-11T14:22:07ZInvestigation of the influence of phase variable restriction modification systems and lipooligosaccharide epitopes on resistance of Haemophilus influenzae to infection by bacteriophagehttp://hdl.handle.net/2381/40031
Title: Investigation of the influence of phase variable restriction modification systems and lipooligosaccharide epitopes on resistance of Haemophilus influenzae to infection by bacteriophage
Authors: Turkington, Christopher Jason Richard
Abstract: The evolution of ON/OFF switching phase variable loci is presumed to have arisen due to the need for bacterial populations to cope with uncertain environments where selection fluctuates between opposing pressures. One such selection is believed to be the presence of bacteriophage. Bacteriophage can influence bacterial evolution by forcing the development of bacteriophage resistance mechanisms within bacterial populations. However, resistance mechanisms can often come at a cost, such as reduced survival against the immune responses. As such, phase variation may circumnavigate this cost by generating heterogeneous populations containing both resistant and sensitive phenotypes.
This study aimed to investigate the two known phase variable bacteriophage resistance genes in Haemophilus influenzae hsdM and lic2A, which encodes the methyltransferase of a type I restriction modification system and a lipooligosacharide biosynthesis associated glycosyltransferase respectively.
Analysis of the diversity of repeat tract lengths and ON/OFF states of these genes across H. influenza genomes within GenBank revealed that while lic2A was ON in the majority of strains, hsdM was OFF. Thus lic2A may be consistently beneficial while the hsdM benefit is transient. Analysis of samples from patients with COPD, showed that lic2A was ON in the majority of samples, while hsdM may also be ON state in a number of samples.
Although a resistance phenotype could be observed for lic2A, no resistance could be observed for hsdM. Therefore, the dynamics of bacteriophage spread through populations heterogeneous for lic2A was investigated. The heterogeneous populations generated by phase variation reduced bacteriophage dispersal through bacterial populations. This mechanism may also allow bacterial populations to adjust their heterogeneity levels to control bacteriophage densities. The heterogeneity may further create diverse bacteriophage densities across the bacterial macropopulation through resistant populations acting as a barrier.
The results demonstrate the potential for phase variation to aid in the survival of bacterial populations against bacteriophage predation.Mon, 10 Jul 2017 09:19:08 GMThttp://hdl.handle.net/2381/400312017-07-10T09:19:08ZInterrogation of Rab8 as a therapeutic target for Huntington’s disease in Drosophila melanogasterhttp://hdl.handle.net/2381/39924
Title: Interrogation of Rab8 as a therapeutic target for Huntington’s disease in Drosophila melanogaster
Authors: Delfino, Laura
Abstract: Huntington’s disease (HD) is a familial neurodegenerative disorder largely caused by atrophy in the striatal and cortical regions of the brain. At the molecular level HD is triggered by a trinucleotide CAG repeat expansion in the Huntingtin gene (HTT), which encodes a poly glutamine (polyQ) stretch, and ultimately leads to the production of the toxic, aggregationprone protein mutant HTT (mHTT). Upon expression of mHTT, several cellular pathways are either disrupted or impaired, including the vesicle trafficking directed by the Rab GTPase family. Here, I focused on Rab8, a protein involved in the secretory traffic from the trans-Golgi network to the plasma membrane, whose down-regulation has been shown to worsen HDrelated phenotypes in mammalian cells. My results show that pan-neuronal expression of Drosophila Rab8 (dRAB8) provides neuroprotection against HD-relevant phenotypes in Drosophila by reducing degeneration of the eye photoreceptors, ameliorating the rate of fly emergence from the pupal case and increasing average lifespan of adult flies. Notably, this rescue depends on the nucleotide-binding state of the GTPase. The protective role of dRAB8 was also validated in a subset of circadian clock cells, the Pigment Dispersing Factor (PDF) neurons. mHTT triggered arrhythmic locomotor behaviour in constant darkness and progressive death of a cluster of PDF neurons, the small lateral neurons ventral (s-LNvs), phenotypes which were partially or completely rescued by dRAB8 overexpression. The levels of aggregated mHTT are increased upon dRAB8 co-expression in flies and experiments performed in HEK293T cells suggest that the interaction dynamics between mHTT and dRAB8 increase in a polyQ dependent manner. Aggregation has been shown to be neuroprotective against toxic soluble mHTT species in several HD model organisms and might underlie the mechanism of dRAB8 rescue. In summary, this study validates Rab8 as a modifier of HD in Drosophila, provides insight into its mechanism of action, and may ultimately inform novel therapeutic approaches for HD.Tue, 20 Jun 2017 09:29:38 GMThttp://hdl.handle.net/2381/399242017-06-20T09:29:38ZGenomics and population dynamics of phase variable genes in Campylobacterhttp://hdl.handle.net/2381/39872
Title: Genomics and population dynamics of phase variable genes in Campylobacter
Authors: Aidley, Jack Benedict
Abstract: Phase variation is a feature of many pathogenic bacteria, including Campylobacter jejuni – a leading cause of food-borne gastroenteritis. C. jejuni has many phase variable (PV) genes which exhibit high frequency, stochastic, reversible switching between ON and OFF expression states. This results from instability in simple sequence repeats (SSRs).
This study is the first to conduct a widescale survey of the Campylobacter ‘phasome’ (the set of PV genes present in a genome). A new program, PhasomeIt, was developed to identify and compare all SSR-mediated phase variable genes in 77 complete Campylobacter genomes. Surprisingly, there are a large number of rare PV genes with few, or no, homologues in other genomes. These exist alongside a “core phasome” of PV genes associated with particular species. This suggests a significant role for phasome diversity in Campylobacter population and disease dynamics.
SSR tract length influences rates of PV, however it is not known whether differences in PV rate are biologically significant or whether a threshold effect exists. A cyclical assaywas developed which allows alternation of experimental conditions favouring the ON and OFF states of cj1421c. Bacteriophage F336 selects for the OFF state whilst human serum selects for the ON state, and both produce complete selective sweeps. These agents were combined for a complete ON→OFF→ON cycle. Using an in silico model of this assay it was demonstrated that a broad range of conditions favour PV over non-PV genes whilst variation rate is primarily dependent on duration of selection. Extending this modelling approach to the interaction of non-selective bottlenecks and PV loci indicated that smaller bottlenecks have a disruptive effect on population dynamics whilst larger bottlenecks preserve increased diversity. These differences are capable of producing stochastic differences in output populations that may drive host-to-host diversity and result in rare occurrence of disease sequelae.Thu, 08 Jun 2017 15:05:59 GMThttp://hdl.handle.net/2381/398722017-06-08T15:05:59ZElectromagnetic field effects in Drosophila melanogasterhttp://hdl.handle.net/2381/39852
Title: Electromagnetic field effects in Drosophila melanogaster
Authors: Fedele, Giorgio
Abstract: Many higher animals have evolved the ability to use the Earth’s magnetic field, particularly for orientation. However, the biophysical mechanism by which magnetoreception is achieved remains elusive. One theoretical model (the radical pair mechanism - RPM) proposes that the geomagnetic field is perceived by chemical reactions involving the blue-light photoreceptor Cryptochrome (CRY). Recent evidence supports the RPM in Drosophila melanogaster and reveals a mechanistic link with the circadian clock. Here I have confirmed, albeit with rather different results, that a low frequency electromagnetic field (AC-EMF) along with a Static Field (SF) exposure does affect circadian and activity behaviour in the fruit fly. Furthermore, I have developed two new assays to investigate the effects of EMF in Drosophila melanogaster, negative geotaxis and an additional light wavelength preference assay, revealing a net CRY-dependent response. My data support the idea of CRY mediated magnetoreception, thereby indirectly supporting the RPM.
Furthermore, I provide some striking new results that challenge our view that only the canonical clock neurons contribute to behavioural rhythms in Drosophila melanogaster.Tue, 30 May 2017 14:53:20 GMThttp://hdl.handle.net/2381/398522017-05-30T14:53:20ZNasonia Vitripennis: An Emerging Model Organism for Chronobiologyhttp://hdl.handle.net/2381/39828
Title: Nasonia Vitripennis: An Emerging Model Organism for Chronobiology
Authors: Davies, Nathaniel Jude
Abstract: The circadian clock co-ordinates a wide range of biological processes in animals and as such has implications for a range of research topics, including human disease. Recent research has shown that Drosophila, the most widely used insect clock model, may be somewhat esoteric, suggesting that other insects may make more suitable models. The work presented in this thesis advances the jewel wasp Nasonia vitripennis as a model for circadian research. Firstly, the response of the Nasonia circadian clock to temperature was characterised, revealing the presence of a temperature compensation mechanism more strict in constant darkness than in constant light. This work was followed up by the profiling of the circadian transcriptome in Nasonia in both of these constant conditions. This work revealed fundamental differences in the dynamics of circadian transcription between Drosophila and Nasonia, as well as identifying temporal separation of biological function in the wasp, most notably of anabolic and catabolic processes. Secondly, conserved upstream non-coding sequences were identified and analysed, shedding light on the mechanisms of transcriptional and translational control in Nasonia. This analysis revealed conservation of the regulatory elements which control regulatory genes, indicating the presence of conserved regulatory cascades. This work also reports the identification and analysis of conserved regulatory mechanisms in RNA, including conserved secondary structures, carrying implications outside of chronobiology. Thirdly and finally, a gene-focused database was created for Nasonia to facilitate such research. Genome annotation data was processed and combined with functional data from published RNA-seq and microarray datasets, along with the original analysis of a genome-wide DNA methylation dataset, and the implementation of several tools.Mon, 22 May 2017 11:59:27 GMThttp://hdl.handle.net/2381/398282017-05-22T11:59:27ZMolecular characterization of the Parkinson’s associated protein DJ-1http://hdl.handle.net/2381/39640
Title: Molecular characterization of the Parkinson’s associated protein DJ-1
Authors: Hassanjani, Mahdieh
Abstract: Mutations in DJ-1 (PARK7), a conserved protein of 189 amino acids, cause autosomal recessive cases of Parkinson’s disease (PD). DJ-1 appears to play a central role in protecting cells from oxidative stress, which likely has relevance for its role in PD pathogenesis. Biochemical and crystallographic approaches indicate that DJ-1 dimerizes, which is likely fundamental for its stability and normal function. A main focus of this thesis was identifying and characterizing DJ-1 dimerization modifiers through an unbiased screen of a kinase and phosphatase inhibitor library, taking advantage of bimolecular fluorescence complementation (BiFC) as a readout for DJ-1 dimerisation in living cells. To address this aim, we generated HEK 293T cell clones stably over-expressing DJ-1 BiFC constructs and optimised high throughput Cell^RScan^ R screening. This approach identified two kinase inhibitors (Bosutinib and KW2449) which decrease DJ-1 dimerisation in an oxidative stress-dependent manner. Furthermore, to indicate whether or not the observed effects of the kinase inhibitors on DJ-1 dimerization were due to a direct alteration of DJ-1 phosphorylation status or were indirect effects, we studied DJ-1 phosphorylation at all potential sites on its dimerization and stability by generating phosphomimic and phosphoblocking mutants. This work indicates that phosphorylation of key residues of DJ-1 likely dramatically reduces its stablilty. Additionally, by using the BiFC approach we found a direct interaction in living cells of DJ-1 with microtubule-associated protein tau, which is known to be hyper phosphorylated and aggregated in neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease. These analyses suggest that alterations in DJ-1 dimerization, stability and phosphorylation status in normal conditions and in response to oxidative stress may shed more light on DJ-1 function and its role associated with PD pathogenesis.Mon, 10 Apr 2017 15:30:29 GMThttp://hdl.handle.net/2381/396402017-04-10T15:30:29ZDeciphering the molecular mechanisms underlying the photoperiodic clock in the parasitic wasp, Nasonia vitripennishttp://hdl.handle.net/2381/39537
Title: Deciphering the molecular mechanisms underlying the photoperiodic clock in the parasitic wasp, Nasonia vitripennis
Authors: Flavell, Laura
Abstract: Nasonia vitripennis is an emerging insect model, with great potential in the field of biological rhythms and seasonality. Nasonia possess a robust a response to photoperiod, in which adult females exposed to short winter-like days generate progeny that will undergo a developmental arrest (diapause). The molecular basis underlying the photoperiodic clock is unknown. We expect this response to be underpinned by changes in gene expression between long (LP) and short (SP) photoperiod exposed wasps. To identify candidate genes, I employed RNA sequencing (RNAseq) to profile the transcriptome of the wasp head in both conditions. This identified 66 transcripts as being significantly differentially expressed (FDR < 0.05). Utilising RNAi, select candidate genes were knocked down in the wasp by injecting dsRNA into pupae. The ability of the wasps to respond to photoperiod (compared to a control group injected with dsRNA against GFP) was assessed. Wasps injected with jhamt dsRNA responded more rapidly to SP conditions with an elevated level of diapause in both photoperiods, while both obp03b and ggt-injected wasps showed no change in response in LP but displayed significantly reduced levels of diapause in SP. These results suggest a causal role for these genes in the photoperiodic response. In a related set of experiments, I have expanded the current knowledge of miRNA composition in the wasp and assessed for the potential role of miRNA in photoperiodism. Through small RNA sequencing, I profiled the changes in miRNA expression in the adult wasp head in response to photoperiod, experimentally validated currently predicted miRNA and predicted a further 106 potential miRNA previously unknown in the wasp. I also explored the potential of maternally deposited miRNA as a mechanism of the transfer of photoperiodic information, as differentially expressed miRNA were observed in young Nasonia embryos, prior to the start of transcription from the zygotic genome. This thesis promotes the use of a new model organism and presents an account of transcriptional and post-transcriptional regulation of gene expression related to seasonal timing in Nasonia vitripennis.Tue, 21 Mar 2017 16:48:22 GMThttp://hdl.handle.net/2381/395372017-03-21T16:48:22ZPhenotypic and Genotypic Investigation of Persistent Staphylococcus aureus Bacteraemia Isolateshttp://hdl.handle.net/2381/39405
Title: Phenotypic and Genotypic Investigation of Persistent Staphylococcus aureus Bacteraemia Isolates
Authors: Richards, Rebecca
Abstract: Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) remain a serious clinical threat and a leading cause of healthcare- and community-associated infections. Blood stream infections or bacteraemia caused by S. aureus are further complicated by the phenomenon of bacterial persistence and treatment failure despite confirmed in vitro susceptibility of the infecting strain to the administered antibiotics. Moreover, it is unclear how S. aureus evades the host immune system for the prolonged duration of a persistent infection. This study aimed to answer these two clinically relevant, biological questions by characterising multiple persistent and resolved MRSA bacteraemia isolates with the view to identifying persistent isolate associated phenotypic and genotypic traits, and novel mechanism(s) for persistence development.
From this work, two distinctly novel S. aureus persistence mechanisms are presented. The first involved in vivo strain evolution induced by antibiotic exposure (daptomycin) during two independent incidences of persistence (PB1 and PB3), resulting in mprF gain-of-function mutations in the persisting bacterial population. This led to daptomycin non-susceptibility and the emergence of novel persistence associated virulence phenotypes, which included a nutrient deprived growth adaption, increased immune evasion, adhesion and stress response associated surface proteins, and attenuated virulence. The second mechanism presented (PB2 and PB5) did not display any defining persistence associated traits; however these bacteraemia were not treated with daptomycin, which further supports the daptomycin induced, MprF mediated persistence mechanism presented for the PB1 and PB3 cases.
This study is the first of its kind to investigate multiple persistent MRSA bacteraemia inclusive of numerous temporally spaced infective isolates, in parallel with contemporaneous resolved bacteraemia isolates of the same genetic background. The subsequent findings of this study have massive implications for the current clinical regimes implemented during complex S. aureus bacteraemia, particularly antibiotic treatment choices, and potentially for other cases of bacterial infection where persistence is frequently observed.Tue, 07 Mar 2017 15:18:57 GMThttp://hdl.handle.net/2381/394052017-03-07T15:18:57ZNuclear and chloroplast genome diversity in apomictic microspecies of Taraxacumhttp://hdl.handle.net/2381/39395
Title: Nuclear and chloroplast genome diversity in apomictic microspecies of Taraxacum
Authors: M. Salih, Rubar Hussein
Abstract: Whole genomic survey sequences were obtained for Taraxacum obtusifrons Markl. (O978); T. stridulum Trávniček ined. (S3); and T. amplum Markl. (A978), three apomictic triploid (2n=3x=24) dandelions from the T. officinale agg. (Asteraceae) Retroelement-based markers and chloroplast data showed that S3 and O978 are genetically the most similar microspecies. Genomic diversity in Taraxacum and also Hieracium was high, discriminating species but not showing phylogeny; major groups of retroelements were abundant in both genera. The chloroplast genomes of accessions O978 and S3 were identical. Repetitive DNA including transposable elements (TEs) are dynamically evolving in genomes, but their variability and abundance make them challenging to study using molecular biology. In the current study, we used the whole genomic sequences to investigate the repetitive structure, diversity and components of the three closely related Taraxacum accessions. Analysis of about 45Gb sequence (10x to 20× genome coverage) of three closely related Taraxacum microspecies, were analysed by graph-based clustering of the raw reads (using the program RepeatExplorer) and frequency analysis of all DNA motifs possible for various motif lengths (k-mer analysis). Different DNA motif lengths were evaluated and complemented the graph-based results. Graph-based clustering showed that many of the Taraxacum microspecies repeats consist of Ty1-copia (13-16%) and Ty3-gypsy (10-14%) family retroelements, while DNA transposons were rare. Unclassified repetitive DNA sequence clusters were investigated. In situ hybridization was used to localize major repetitive DNA families on chromosomes. Apart from 5S and 45S rDNA and telomere sequences, few tandemly repeated DNA motifs were found, although a 49bp repeat was found at some centromeres. There were differences between the three Taraxacum microspecies in genomic proportions and locations for repetitive DNA types suggesting many sequence motifs are evolving rapidly with increasing or decreasing copy numbers. A class of repetitive DNA has been recognized as Passively Amplified DNA Sequences, PADS.Mon, 06 Mar 2017 11:44:20 GMThttp://hdl.handle.net/2381/393952017-03-06T11:44:20ZSystematic and genomic studies in the genus Aubrieta (Brassicaceae)http://hdl.handle.net/2381/39393
Title: Systematic and genomic studies in the genus Aubrieta (Brassicaceae)
Authors: Muhammed, Jotyar Jassim
Abstract: The study focuses on the herbaceous perennial plant genus Aubrieta (Brassicaceae). Distributed from Armenia through the Levant and Anatolia to Greece, the Balkans and Italy, the species have proved difficult to distinguish. About 12 species are currently recognised, although estimates range from only one up to about 20 or more. Furthermore, their evolutionary relationships are unknown. In order to remedy this situation molecular and cytogenetical studies were conducted.
Next Generation Sequencing (NGS) data were produced to generate a complete chloroplast genome for four species of Aubrieta in order to confirm the phylogenetic position of the genus in the family. Earlier suggestions that it belongs in tribe Arabdieae were confirmed. Details of plastome structure were analysed in A. gracilis, which was shown to have 88 protein-encoding, 37 transfer-RNA and eight ribosomal-RNA genes.
A phylogenetic analysis of the genus was conducted based on chloroplast and nuclear sequences as well as mitochondrial RFLPs. Five chloroplast regions (matK, trnD-trnT, ycf6-psbM, rps11-rpl36 and trnH-psbA) and two nuclear genes (duo1 and rbp2) were amplified and sequenced successfully. Six mitochondrial gene regions (Orf114, Nad9-ccmFN2, Orf25, matR, ccm FC, trnK-rps3) were studied by means of restriction enzymes. Data analyses show that Aubrieta comprises the annual, pan-Mediterranean Arabis verna plus perennial taxa that fall into one of five geographically delimited gene pools: i) Near East (Levant, Iraq and Iran); ii) Anatolia; iii) Aegean Basin; iv) Greece, Albania, Bulgaria (Pindus Mts and associated ranges); and v) Trans-Adriatic Sea and Sicily. There was some disagreement between the plastid and nuclear trees, which was attributed to hybridisation, chiefly affecting the taxa occupying the Aegean Basin.
Evolution in the genus appears to have proceeded largely at the diploid level (2n=2x=16). In order to see what changes at the chromosome level have accompanied speciation, fluorescence in situ hybridization (FISH) studies were conducted. The probes pTa71 and pTa794 were used to locate the position of 45S and 5S rDNA sites on the chromosomes. The number of 45S rDNA sites are 2, 4, 5, or 6, localized on short-arms, the centromere and on long-arms. The number of 5S rDNA sites is a constant two, located either on short-arms or long-arms. These rDNA sites (45S and 5S) are either located on different chromosomes or shared by one or two chromosomes. Speciation is accompanied (promoted?) by translocations and duplications. Hybridisation was confirmed in the genus.
The timing of the various bifurcations in the evolutionary tree were estimated from a study of the concatenated chloroplast sequences, but the major split into an annual lineage (Arabis verna) and a perennial lineage appears to date from 1.4 Mya. The hybridisation events involving the Aegean Basin taxa appear to date from the early to mid-Pleistocene (ca 600-800 Kya), a time when considerable parts of the Aegean were above sea level.
The taxonomy of the genus is still problematic, it being impossible to diagnose the five geographical genepools by means of morphological characters. Instead, a splitting approach is adopted whereby regional or local phenotypes are recognised as species. This can be unsatisfactory in some cases where there is considerable morphological, but not geographical, overlap. A total of 21 species, including Arabis verna which is recombined into Aubrieta, is recognised.Mon, 06 Mar 2017 11:18:42 GMThttp://hdl.handle.net/2381/393932017-03-06T11:18:42ZEpigenetic Mechanisms Underlying Seasonal Timing in Nasonia vitripennishttp://hdl.handle.net/2381/39221
Title: Epigenetic Mechanisms Underlying Seasonal Timing in Nasonia vitripennis
Authors: Bafna, Akanksha
Abstract: Many organisms monitor the annual change in day-length, and use this information for seasonal timing of their developmental, physiological and behavioural response. The molecular mechanisms underlying this photoperiodic timing are largely unknown. The wasp, Nasonia vitripennis, is an emerging model organism that exhibits a strong photoperiodic response: short autumnal days experienced by females leads to the inductionof developmental arrest (diapause) in their progeny, allowing winter survival of the larvae. How do the females control the developmental trajectory of their offspring is unclear. Here, I took advantage of the available complete genome sequence of the wasp, and tested the role of epigenetics in the photoperiodic response. I used reduced representation bisulfite sequencing (RRBS) to profile DNA methylation in adult females subjected to different photoperiods, and identified substantial differential methylation at the single base level. I have also found that knocking-down DNAmethyltransferase (Dnmt1a, Dnmt3), or blocking DNA methylation pharmacologically, largely disrupts thephotoperiodic diapause response of the wasps. In another set of experiments, I assessed the prevalence of 5-hydroxy methyl cytosine (5hmC), which is an intermediate in DNA demethylation in mammals. The results show that 5hmC is present in Nasonia although in limited amount and suggests that 5hmC-dependent demethylation may be evolutionary conserved in invertebrates. The role of microRNA (miRNA) in the photoperiodic response was also tested. I experimentally validated 32 of the computationally predicted Nasonia miRNA and tested their expression levels by using stem-loop real-time PCR. I identified significant differential expression in a sub-set of miRNA, which was induced by the photoperiod. To my knowledge, this is the first example uncovering the role of epigenetics in photoperiodic timing in insects.Fri, 20 Jan 2017 10:13:45 GMThttp://hdl.handle.net/2381/392212017-01-20T10:13:45ZThe Effects of Air Pollution on Respiratory Bacteriahttp://hdl.handle.net/2381/39149
Title: The Effects of Air Pollution on Respiratory Bacteria
Authors: Hussey, Shane J. K.
Abstract: Particulate Matter (PM), a major component of air pollution, is associated with a
variety of cardiorespiratory diseases including acute lower respiratory tract infections.
It is well established that PM has detrimental effects on the host, causing tissue
damage, oxidative stress, and modulating the immune system. However there has been
extremely limited research into the effects of PM on bacteria, the organisms responsible
for the respiratory infections associated with PM exposure.
This project investigated whether Black Carbon (BC), a major component of PM
produced as a by-product of fossil fuel combustion, directly affects respiratory tract
bacteria. Two model opportunistic pathogens of the respiratory tract were chosen for
this investigation, Streptococcus pneumoniae and Staphylococcus aureus.
BC was found to alter biofilm formation, structure, matrix composition, and
functioning, of both S. pneumoniae and S. aureus, as well as inhibiting planktonic
growth. Interestingly, these effects were strain-dependent. Furthermore, BC promoted
dissemination of S. pneumoniae from the nasopharynx to the lower respiratory tract in
an in vivo murine colonisation model. BC was not observed to alter the respiratory tract
microbiota in this project, however a variety of limitations which may have prevented a
definitive conclusion being reached are presented.
This study provides the first evidence to show that bacteria are directly affected by PM,
and thereby suggests that the adverse health effects of PM may not only be due to
effects on host tissues, but that modulation of bacterial behaviour may also have a role.
The findings of this study therefore show the potential importance of this overlooked
field.Mon, 16 Jan 2017 11:37:12 GMThttp://hdl.handle.net/2381/391492017-01-16T11:37:12ZPhenotypic Consequences of β-Defensin Copy Number Variation in Humanshttp://hdl.handle.net/2381/38814
Title: Phenotypic Consequences of β-Defensin Copy Number Variation in Humans
Authors: Abujaber, Razan
Abstract: Beta defensins (DEFB) at the 8p23.1 genomic location are multifunctional secreted short peptides that have antibacterial and antiviral action in many species and possess immune cell signal activity, constituting a link between innate and adaptive immunity. In humans, the β-defensin region is known to be copy number variable (CNV) and contains seven genes repeated as a block, with a diploid copy number between 1 and 12.
This thesis shall explore the structural variability of the β-defensin CNV region; compare and contrast the different methods used for calling DEFB CNVs and investigate the role of CNVs of DEFB in various diseases. One of its aims is to also develop a model system to investigate if DEFB expression levels differ with CN in response to treatment with Pneumolysin by using Normal Human Bronchial Epithelial (NHBE) cells.
Results from this thesis confirm that the DEFB CNV region is 322kb in length, with a polymorphic inversion that occurs at a prevalence of 30% at the 8p23.1 genomic location that is independent of the DEFB CN. Paralogue Ratio Test (PRT) proved to be the best method of genotyping DEFB CNV especially in larger cohorts. In addition, work from this thesis also founded the basis of developing an in vitro model system to investigate whether DEFB expression levels differ with CN in response to treatment with pneumolysin by using Normal Human Bronchial Epithelial (NHBE) cells. As far as case/control and cohort studies are concerned, results from this thesis show that DEFB CN is not associated with lung function in the general population and has no effect on patients with COPD and Asthma, nor does it support previous results that present an association between HIV viral load and DEFB CN. DEFB CN was also found not to be associated with recurrent UTIs in VUR patients, nor with hypertension. Data suggested that DEFB CN might be associated with BMI but this has not been reproduced in a smaller cohort.Mon, 05 Dec 2016 11:49:31 GMThttp://hdl.handle.net/2381/388142016-12-05T11:49:31ZAdvancing molecular crustacean chronobiology through the characterisation of the circadian clock in two malacostracan species, Euphausia superba and Parhyale hawaiensishttp://hdl.handle.net/2381/38216
Title: Advancing molecular crustacean chronobiology through the characterisation of the circadian clock in two malacostracan species, Euphausia superba and Parhyale hawaiensis
Authors: Hunt, Benjamin James
Abstract: The ability to entrain to environmental cycles and therefore anticipate and prepare for the changes they predictably bring is the preserve of the endogenous biological clock, most widely studied at the circadian level. Despite a rich history of research into the behavioural and physiological rhythms shown by many crustacean species, the underlying molecular system driving such traits is not well understood. The aim of this research was to develop our understanding of crustacean clocks through the study of two species, one of major ecological importance and the other a powerful model organism.
The Antarctic krill Euphausia superba is a keystone species in the Southern Ocean ecosystem, and evidence suggests that the clock may influence both daily and seasonal rhythms. Using a variety of approaches, including the creation of a de novo assembled head transcriptome, a full suite of clock-related genes have now been cloned and characterised. Unlike many species Euphausia superba possesses orthologs of every canonical core clock gene, and cell culture assays indicate that the central feedback loop has the capacity for complete transcriptional inhibition via two separate pathways, raising the possibility that the krill clock may be an ancestral type or employ multiple oscillators to control rhythms of differing periods.
In contrast to the relatively intractable krill, the amphipod Parhyale hawaiensis has simple maintenance requirements and an extensive genetic toolkit with the potential to enable sophisticated dissection of the molecular clock. With the aim of laying the groundwork for future research the clock genes of this species have also been identified, along with the development of a locomotor activity assay. Parhyale hawaiensis shows evidence of bimodal patterns of activity under the control of a molecular clock that combines mammalian-like characteristics with some unique features worthy of further investigation.Mon, 17 Oct 2016 12:07:34 GMThttp://hdl.handle.net/2381/382162016-10-17T12:07:34ZRegulation of Telomere Length and its Maintenance in the Absence of Telomerase in Budding Yeast Saccharomyces Cerevisiaehttp://hdl.handle.net/2381/38124
Title: Regulation of Telomere Length and its Maintenance in the Absence of Telomerase in Budding Yeast Saccharomyces Cerevisiae
Authors: Aryal, Usha
Abstract: Telomeres are ribo-nucleoprotein structures that cap the end of linear chromosomes protecting them from nucleolytic degradation. In budding yeast Saccharomyces cerevisiae, telomeres are maintained by telomerase and a network of over 300 telomere length maintenance genes. Loss of telomerase leads to progressive shortening of telomeres and eventually cellular senescence. However, a few cells can undergo RAD52 dependent recombination to elongate telomeres and become ‘survivors’. Survivors can be of two major types; Type I and Type II, characterized by amplification of the sub-telomeric Y’ repeats or the telomeric TG₁₋₃repeats respectively.
In this study, I have demonstrated that in addition to the genetic components, initial telomere length and the timing of senescence are essential factors that influence telomerase negative survival. Longer initial telomere lengths favour Type II survivor pathway, lead to increased efficiency of recovery from crisis and higher overall frequency of survivor formation. Furthermore, longer initial telomeres increased the proportion of Type II and Type II-like survivors in the absence of RAD59, a gene generally required for Type II survival. Early senescence was induced from a single critically short telomere in a telomerase negative cell population and led to higher proportion of Type II survivors. Contrary to previous assumptions, the increased proportion of Type II survivors observed was independent to the telomere length at the time of senescence.
Genetic variants that regulate telomere lengths were explored via inter-cross QTL (i-QTL) Multipool analysis using parents from two independent inbred natural populations of S. cerevisiae. Seven candidate genes with known telomere function were identified, with the most prominent QTLs being EST2 and STN1. Five intervals containing genes with no prior association to telomere length regulation were also identified. This study demonstrated the sensitivity of the i-QTL Multipool method in determining novel genetic variants regulating telomere length even with a small sample size.Tue, 04 Oct 2016 09:44:17 GMThttp://hdl.handle.net/2381/381242016-10-04T09:44:17ZThe influence of ploidy-specific expression on selectionhttp://hdl.handle.net/2381/38035
Title: The influence of ploidy-specific expression on selection
Authors: Harrison, Mark Christian
Abstract: More efficient selection is expected for haploid-expressed genes compared diploidexpressed
genes. This is because recessive mutations can be masked from selection
by a dominant allele in diploids but are always exposed to selection in haploids. The
significance of this effect for haplodiploids was recognised by White in 1945, who
predicted less efficient selection on genes with diploid-limited expression.
I present the first empirical support for these predictions for haplodiploids on
the buff-tailed bumblebee, Bombus terrestris. I found evidence for weaker purifying
selection on diploid-biased genes compared to haploid-expressed genes. This has led
to higher protein divergence rates and polymorphism levels in diploid-biased genes
compared to haploid-expressed genes. In contrast, I found no evidence of greater
positive selection on haploid-expressed genes, suggesting that most new, recessive
mutations may be deleterious.
In a second experiment I tested the effect of ploidy-specific selection in the plant
Arabidopsis thaliana by comparing selection patterns between haploid pollen genes
and diploid sporophytic genes. I detected evidence for a change in selection patterns
possibly due to a loss of self-incompatibility. Divergence data indicate stronger
positive selection within pollen genes during a period dominated by outcrossing,
likely caused by pollen competition and haploid selection. Polymorphism data, on
the other hand, reveal signs of relaxed selection within pollen genes, possibly due
to high homozygosity levels, which reduce pollen competition and the masking of
recessive mutations in diploid genes.
In a third study I used the data produced for determining ploidy-biased genes in
B. terrestris to infer expression patterns involved in caste determination. This is the
first broad scale analysis on caste determination in bumblebees. One major finding
was that the expression patterns of bumblebee workers more closely resemble those
of queens when reproductive compared to higher eusocial Hymenoptera, possibly
due to the more plastic nature of bumblebee worker castes.Fri, 09 Sep 2016 11:28:47 GMThttp://hdl.handle.net/2381/380352016-09-09T11:28:47ZFunctional and Localization Studies of Human Kynurenine 3-Monooxygenasehttp://hdl.handle.net/2381/37835
Title: Functional and Localization Studies of Human Kynurenine 3-Monooxygenase
Authors: Swaih, Aisha Mahmod O.
Abstract: Kynurenine 3-monooxygensae (KMO) is an outer mitochondrial membrane protein which plays a critical regulatory role in the kynurenine pathway (KP), catalysing the production of 3-hydroxykynurenine (3-HK). Increased KMO activity likely contributes to the excitotoxicity seen in neurodegenerative disorders by elevating the levels of the neurotoxic KP metabolites 3-HK and quinolinic acid. Studies employing models of Huntington’s disease (HD) have shown that inhibition of KMO is neuroprotective, making KMO a potential therapeutic target for this disorder. This study interrogates the subcellular localisation of human KMO and dissects the interaction between KMO and the huntingtin (HTT) protein, mutations in which cause HD.
Confocal microscopy based co-localisation studies of KMO demonstrated that full length KMO (flKMO) was exclusively localised to the mitochondria when expressed in HEK293T cells. Notably, deleting a C-terminal portion of flKMO which contains a putative transmembrane domain mis-localised the remaining protein (tKMO) to other cellular compartments. Localization of flKMO to the outer mitochondrial membrane was further confirmed via transmission electron microscopy.
To study potential interactions between flKMO and HTT in living cells, bimolecular fluorescence complementation (BiFC) assay was utilised, which is based upon reconstitution of split fluorescence proteins. The BiFC approach allowed visualisation and quantification of flKMO interaction with both WT HTT and soluble mHTT fragments at the mitochondria. The strength of this interaction is inversely correlated to the length of the HTT polyglutamine expansion. Increased mitochondrial sub-cellular localisation of BiFC HTT constructs was confirmed via microscopy. tKMO however did not interact with HTT via the BiFC system, indicating that the C-terminal region of flKMO is important for both mitochondrial localisation and protein interaction. In total, these data suggest that flKMO-HTT interactions at the mitochondria may be biologically significant and could play a role in regulating KMO activity, and that in HD this regulatory process is impaired.Wed, 13 Jul 2016 15:17:41 GMThttp://hdl.handle.net/2381/378352016-07-13T15:17:41ZThe Effects of Extremely Low-Frequency Magnetic Fields on Mutation Induction in Micehttp://hdl.handle.net/2381/37610
Title: The Effects of Extremely Low-Frequency Magnetic Fields on Mutation Induction in Mice
Authors: Wilson, James William
Abstract: Extremely low-frequency magnetic fields (ELF-MF) are classified as possibly carcinogenic, despite inconsistent data and no plausible biological mechanism linking their universal exposure with childhood leukaemia and genotoxic effects.
Given discrepancies in mutagenic data and widespread public concern over genotoxic effects, this study was designed to provide an in-depth analysis of potential molecular changes induced by ELF-MF exposure in vivo. Seven-week old, BALB/c x CBA/Ca hybrid F1 male mice were exposed to 50 Hz magnetic fields of 10, 100 and 300 μT for 2- or 15 hours. Blood and sperm DNA samples were collected 12 weeks post-exposure and mutation induction frequencies established at the Expanded Simple Tandem Repeat (ESTR) Ms6-hm loci using single-molecule PCR (SM-PCR). Likewise, Ms6-hm mutation induction frequencies were established in age-matched sham-treated hybrid males (control group) and those exposed to 1 Gy acute X-rays (positive controls).
No significant increases in ESTR mutation frequencies were detected in either tissue at any ELF-MF exposure parameter compared to their sham-treated controls. Whilst a marginally significant increase was observed in the mutation induction frequency of pooled sperm data, these data should be regarded cautiously due to the lack of correlating dose-dependent responses. Conversely, germline and somatic ESTR mutation frequencies were significantly elevated in males exposed to acute 1 Gy X-rays.
These data were validated in a high-throughput microarray pilot study, whereby no significant alterations in gene expression in kidney cells of hybrid males were detected following ELF-MF exposure. In contrast, five transcripts were significantly up-regulated in the irradiated males.
Ultimately, these findings indicate that, within the analysed range of doses, the in vivo effects of ELF-MF exposure on mutation induction and gene expression are likely to be negligible. This study represents the first methodical attempt to determine mutation frequencies in vivo after continuous exposure to 50 Hz ELF-MFs up to 300 μT.Thu, 19 May 2016 14:39:12 GMThttp://hdl.handle.net/2381/376102016-05-19T14:39:12ZElectrophysiological characterisation of Drosophila central clock neurons in health and diseasehttp://hdl.handle.net/2381/37465
Title: Electrophysiological characterisation of Drosophila central clock neurons in health and disease
Authors: Nugent, Marie Louise
Abstract: The importance of normal circadian clock function in maintaining good overall health has become widely accepted. Daily rhythms in physiology and behaviour are driven by a complex circuit of circadian neurons, of which in Drosophila remains an elusive part of circadian control. A study published in 2011 revealed the ability of CRY to behave as a neuronal activator in light conditions, eliciting an increase in firing frequency when activated; possibly via the modulation of potassium channels in the cell membrane using whole cell patch clamp. The use of this technique in Drosophila CNS neurons is limited due to difficulties in obtaining and interpreting the data generated. This project aims to contribute to the field via exploring its use and limitations, as well as dissect the role of blue light photoreceptor CRYPTOCHROME within the circadian clock neuron circuit by manipulating normal levels of CRY. This was achieved by using a constitutively active from of CRY, CRYΔ, a cry knockout, cry0 and compared these to controls with undisturbed levels of CRY. We also considered the impact of these changes depending on the time of day by recording l-LNv physiology in the morning and the evening. This study revealed that CRY modulates potassium channels throughout the day to support the circadian-regulated changes in l-LNv physiology, of which some are already known such as firing frequency and resting membrane potential. These changes have suggested that along with CRY’s instant ability to influence potassium channels in response to light input in the morning, a delayed signalling mechanism that impacts potassium channels in the evening could occur.
It is well established that clock degradation occurs as an early symptom in many neurodegenerative diseases, such as Parkinson’s disease. We therefore studied the effect of α-Synuclein expression in l-LNv neurons throughout the process of aging. Interestingly, we have revealed that the process of aging influences aspects of normal l-LNv physiology. In conjunction, α-Synuclein expression disrupted these normal aging processes as well as affect firing frequency and potassium channels outside of this. Although more work is needed to consolidate these findings, this study has provided an insight into clock cell disruption that occurs prior to the loss of neurons. We hope this early signalling disruption could be further investigated to aid earlier diagnosis in the future.Fri, 29 Apr 2016 13:03:42 GMThttp://hdl.handle.net/2381/374652016-04-29T13:03:42ZThe role of transducer-like proteins in Campylobacter jejunihttp://hdl.handle.net/2381/37005
Title: The role of transducer-like proteins in Campylobacter jejuni
Authors: Elgamoudi, Bassam Abusalama
Abstract: Campylobacter jejuni is a significant gastrointestinal pathogen of humans. Many proposed virulence factors in C. jejuni remain poorly characterised, although many studies have already identified chemotaxis as a significant virulence factor. The chemotaxis pathway allows motile bacteria to sense and migrate toward or away from different environmental signals. Transducers like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable this pathogen to respond to changing nutrient concentrations in the intestinal environment through mediating taxis toward or away from chemoeffector stimuli.
In spite of recent research to characterise the role of Tlps in chemotaxis in C. jejuni, these proteins are still not fully understood, in particular, the cytoplasmic group C Tlps (Tlp 5, 6, and 8) which have not been extensively investigated. Here, the role of group C Tlps of C. jejuni NCTC 11168 in chemotaxis, and biofilm formation were investigated. Also the localisation of those proteins was determined. The genes encoding Tlp 5, 6 and 8 were successfully mutated and complemented. Inactivation of tlps showed important phenotypes associated with motility, chemotaxis and biofilm formation. The tlp5-, Δtlp6, tlp8- mutants all showed an altered phenotype in the swarm assay. The tlp5- and tlp8- mutants swarmed significantly further than the wild-type motile-variant, whereas the Δtlp6 mutant showed no differences in spreading on plates. No significant growth differences were seen between mutants, complements and wild-type.
The modified Hard-Agar Plug (t-HAP) assay and μ-slide chemotaxis assay were developed to improve the determination of ligand specificities of Tlp chemoreceptors. The t-HAP assay showed a promising quantitative result which can be used to compare different concentrations of chemoattractant in a short time. Data derived from the t-HAP assay indicated that mutation of tlp 5, 6 and 8 has a significant effect on the chemotactic response to L-serine, L-aspartate and L-proline compared to wild type. The mutant phenotypes in the μ-slide chemotaxis assay correlated with the result of the t-HAP assay, where both show that C. jejuni has preferential chemotactic patterns, with L-serine more preferred to L-proline, L-glutamate and lastly L-aspartate. The mutation of tlp5 and tlp6 change the chemotaxis patterns to L-serine and L- aspartate compared to wild type, which may be related to the adaptation of the methylation status involved in the response to chemoattracts. The tlp8- mutant showed less chemotactic response to all amino acids tested in this study compared to wild type which may relate to a link with energy taxis.
For the first time the localisation of Tlps in C. jejuni using a fluorescent reporter, iLOV, has been examined where the iLOV system was expressed using different promoters. Tlp5 and Tlp8 were shown to be localised at the cell poles. In addition, this is the first report to demonstrate that c-di-GMP-responsive adaption is present in C. jejuni and that there is a link between biofilm dynamics and the level of c-di-GMP. Tlp8 was named CbdA for Campylobacter biofilm dispersion as it was found to be involved in the modulation of c-di-GMP levels to mediate biofilm dispersion. Biofilm dispersion in C. jejuni was also found to be responsive to D-amino acids.
The findings in this thesis indicate that transducer like proteins (group C) have an important role not only in chemotaxis but also in biofilm formation, which may provide a basis for understanding the pathogenicity of this pathogen.Wed, 09 Mar 2016 10:19:58 GMThttp://hdl.handle.net/2381/370052016-03-09T10:19:58ZEvolutionary studies in subtribe Reynoutriineae (Polygonaceae). With contributions to the study of hybridisation in Helosciadium and Berula (Apiaceae) included as appendices.http://hdl.handle.net/2381/36207
Title: Evolutionary studies in subtribe Reynoutriineae (Polygonaceae). With contributions to the study of hybridisation in Helosciadium and Berula (Apiaceae) included as appendices.
Authors: Desjardins, Stuart David
Abstract: Subtribe Reynoutriineae is a monophyletic group within the Polygonaceae, containing three genera: Reynoutria, Muehlenbeckia and Fallopia. The clade is strongly supported by molecular data and is characterised by the presence of a nectariferous gland at the base of leaf petioles, which secretes a sweet, sugary exudate. In this thesis the evolutionary relationships within the subtribe were investigated, and a revised classification is presented that amalgamates all three genera under Fallopia, which has priority. This treatment is supported by phylogenetic analysis, and the detection of intergeneric hybridisation between Reynoutria and Muehlenbeckia.
A phylogenetic analysis of molecular sequence data from six gene regions (two nuclear & four plastid) was conducted with the widest sampling of ingroup taxa for this clade to date, in particular being the first to include species from Fallopia sect. Parogonum. The results of this analysis revealed four main clades within the subtribe: a Reynoutria clade, a Fallopia sect. Parogonum clade, a Fallopia s.s. clade (including Fallopia sects. Fallopia & Sarmentosae), and a Muehlenbeckia clade. The Fallopia s.s. and Muehlenbeckia clades are sister to one another, while the Fallopia sect. Parogonum clade is immediately basal. Fallopia, as currently circumscribed, appears to be paraphyletic as species of Muehlenbeckia are nested within it.
The parentage of putative hybrids collected as open-pollinated seed from Japanese knotweed s.l. (Reynoutria) in New Zealand, where Muehlenbeckia was the nearest identifiable pollen source, was investigated using: chromosome counts, fluorescent in situ hybridisation with labelled total genomic probes (GISH), and sequence data from three gene regions, one plastid (matK) and two nuclear (the ITS & LEAFYi2). The hybrids were found to be the result of intergeneric hybridisation between Reynoutria and Muehlenbeckia, which supports the amalgamation of the genera.
The diversity and geographical origin of Japanese knotweed s.l. in Australasia was also investigated, using: morphology, chromosome counts and PCR-RFLP derived chloroplast haplotypes. The majority of clones match those found in Europe, but there is evidence for an independent introduction from Japan.Thu, 07 Jan 2016 14:16:37 GMThttp://hdl.handle.net/2381/362072016-01-07T14:16:37ZMolecular cytogenetics and genomics of novel wheat-Thinopyrum bessarabicum recombinant lines carrying intercalary translocationshttp://hdl.handle.net/2381/36195
Title: Molecular cytogenetics and genomics of novel wheat-Thinopyrum bessarabicum recombinant lines carrying intercalary translocations
Authors: Patokar, Chetan
Abstract: The diploid wild grass Thinopyrum bessarabicum (2n = 2x = 14, JJ or EbEb) is a rich
source of important genes for bread wheat (2n = 6x = 42) improvement because of its
salinity tolerance and disease resistance. Development of wheat–Th. bessarabicum
translocation lines by backcrossing amphiploids in the absence of the Ph1 gene
(allowing intergenomic recombination) enables its practical utilization in wheat
improvement. Using genomic in situ hybridization (GISH) and repetitive probes for
fluorescent in situ hybridization (FISH), six novel wheat–Th. bessarabicum
translocation lines involving different chromosome segments (T4BS.4BL-4JL,
T6BS.6BL-6JL, T5AS.5AL-5JL, T5DL.5DS-5JS, T2BS.2BL-2JL, and the whole arm
translocation T1AL.1JS) were identified and characterized in this study. No background
translocations between wheat genomes were observed. The involvement of 5 of the 7
chromosomes, and small terminal segments of the Th. bessarabicum chromosome arm
were important, contributing to both reduced linkage drag of the derived lines by
minimizing agronomically deleterious genes from the alien species, and high stability
including transmission of the alien segment. All three wheat genomes were involved in
the translocations with the alien chromosome, and GISH showed the Th. bessarabicum
genome was more closely related to the D genome in wheat. All the introgression lines
were disomic, stable and with good morphological characters. The work also generated
a high-resolution karyotype of two accessions of Th. bessarabicum using multiple
repetitive DNA probes for chromosome identification. A complete CS-Th.
bessarabicum amphiploid (2n=8x=56, AABBDDJJ) was used and each individual Jgenome
unambiguously identified. The established karyotype will be useful for the
rapid identification of potential donor chromosomes in wheat improvement programs,
allowing appropriate alien-chromosome transfer. Genotyping-by-sequencing (GBS)
data was collected from the wheat-Th. bessarabicum introgression lines, but the
complexity of the wheat genome and need for further development of data analysis
pathways limited interpretation.Wed, 06 Jan 2016 16:18:53 GMThttp://hdl.handle.net/2381/361952016-01-06T16:18:53ZThe Role of Hfq in S. aureus Gene Regulationhttp://hdl.handle.net/2381/36167
Title: The Role of Hfq in S. aureus Gene Regulation
Authors: Tarrant, Emma J.
Abstract: Staphylococcus aureus is an important opportunistic pathogen, capable of causing a wide range of diseases. This ability to colonise and infect a variety of different tissues is due to the number of virulence factors it can produce. These factors are tightly regulated so they are only expressed when required and allow rapid adaptation to changing environments. In other bacteria the RNA binding protein Hfq is important for growth, resistance to stresses and virulence. Hfq regulation occurs through RNA stability, processing and translation. However the role of Hfq in S. aureus is controversial as conflicting reports on the subject have been published. Preliminary work in our laboratory indicated a role for Hfq in the positive regulation by the DNA binding protein Fur but the mechanisms involved are unknown. Therefore the aim of this study is to identify targets for Hfq regulation and investigate how Fur is involved in this regulation.
This work demonstrates that S. aureus Hfq, along with Fur, has a key role in the resistance to oxidative stress and the regulation of several important virulence genes including the immune evasion factor, Eap. Hfq was found to regulate eap expression at the post transcriptional level. In addition, both Hfq and Fur were found to regulate eap expression transcriptionally, possibly through regulation of sae, an important virulence gene regulator. Fe-Fur was shown to directly bind the sae promoters suggesting direct positive regulation of sae by Fur. But the mechanisms involved in Hfq regulation remain unclear as Hfq did not have a major affect eap or sae mRNA stability. The regulation by Hfq and Fur shows some strain variation indicating that other factors are involved. Therefore Hfq and Fur play key roles in S. aureus virulence regulation. Further understanding of this complex regulatory network may reveal new targets for antimicrobial development to combat this important pathogen.Tue, 05 Jan 2016 15:44:18 GMThttp://hdl.handle.net/2381/361672016-01-05T15:44:18ZTransgenes of the human hypervariable minisatellite MS32.http://hdl.handle.net/2381/35610
Title: Transgenes of the human hypervariable minisatellite MS32.
Authors: Allen, Maxine Johanna.
Abstract: A high rate of mutation at some minisatellite loci can result in exceptional allelic variability. Often, in addition to length polymorphism, sequence variation exists between the minisatellite repeat units in an allele. The interspersion patterns of minisatellite variant repeat units can be determined using MVR-PCR and in this work the allelic hypervariability observed in earlier studies of Ae MS32 locus in Caucasians was shown to extend to Asian alleles. Previous analysis of changes in mutant MS32 MVR-PCR patterns, revealed a polarity of mutation at one end of the repeat array, implicating the possible influence of cis-acting factors on mutation at the MS32 locus. Transgenic mice were created carrying an MS32 transgene to assess for effects of flanking DNA and transgene structure on MS32 mutation processes. Eight separate insertion events were characterised, five single-copy and three multi-copy, and a 'preference' for integration into mouse gamma satellite DNA was observed. Southern blot pedigree analysis and SP-PCR of sperm DNA were performed to assay for mutations at the transgenic loci. Mutation rates at the single-copy loci were 10 to 100-fold lower than at the endogenous human locus. MVR-PCR analysis of SP-PCR recovered transgene 110D mutation events indicated regions of repeat unit duplication, deletion and switching, and a gene conversion event was recovered at the transgene HOC locus. In contrast, mutation rates at the multicopy loci were high (5 - 9.8%) and unusual types of minisatellite mutation including early embryonic events, the frequent loss of a single restriction site and the common deletion of the entire locus were observed. Environmental and genotypic effects on minisatellite mutation were also studied using SP-PCR on DNA from transgene m 110D positive mice irradiated with 60Co, and assaying for minisatellite instability in human cell line DNA with a aberrant mismatch repair phenotype.Thu, 19 Nov 2015 09:14:37 GMThttp://hdl.handle.net/2381/356102015-11-19T09:14:37ZStudies on the control of mitosis in the plasmodium of physarum polycephalum.http://hdl.handle.net/2381/34477
Title: Studies on the control of mitosis in the plasmodium of physarum polycephalum.
Authors: Sudbery, Peter E.
Abstract: Evidence is cited to support the assumption that the time of mitosis and DNA synthesis in Physarum polycephalum is controlled to maintain a constant DNA: mass ratio. Five possible model mechanisms for the control are discussed and analysed to obtain predictions concerning the response to experimentally induced changes in the DNA: mass ratio. Experiments designed to test these predictions showed the following: 1. If mitosis is delayed either by FUdR and uridine treatment or by puromycin treatment the next intermitotic period is equivalently shortened. 2. If a proportion of DNA is destroyed by UV irradiation then, as shown by other workers, subsequent intermitotic periods are shortened. However, it was shown that an intermitotic period cannot be shortened beyond a minimum length and that this minimum length is independent of the growth rate. 3. If the DNA: mass ratio is lowered by UV irradiation by an amount which is not sufficient to cause the next period to be shortened to the minimum length, then only one shortened intermitotic period is required to restore the steady-state DNA: mass ratio. 4. Comparison of the length of the shortened period following UV irradiation with the amount of DNA destroyed agree with the predictions of two of the models considered. 5. If more than 50% of the DNA is destroyed, two successive mitoses can occur in the complete absence of mass increase. It is argued that these results are inconsistent with three of the models which may be eliminated from consideration. Consideration of experimental evidence from other cell types suggest there are difficulties in applying one of the two remaining models to the more general situation.Thu, 19 Nov 2015 08:53:44 GMThttp://hdl.handle.net/2381/344772015-11-19T08:53:44ZFunctional analysis of the promoter of the 3-phosphoglycerate kinase gene of Aspergillus nidulans.http://hdl.handle.net/2381/34476
Title: Functional analysis of the promoter of the 3-phosphoglycerate kinase gene of Aspergillus nidulans.
Authors: Streatfield, Stephen John.
Abstract: Sequence elements contributing to high level expression of the 3-phosphoglycerate kinase (pgk) gene of Aspergillus nidulans have been investigated by the construction of gene fusion vectors using lacZ (?-galactosidase) of Escherichia coli as an assay system, and including the catabolic dehydroquinase (qutE) gene as a selective marker for the transformation of A.nidulans. The importance of targeting these constructs to a specific gene locus has been demonstrated by the analysis of a large number of transformed strains which reveals that the expression of the lacZ reporter gene is dependent upon the site of integration of the vector into the genome, and that when targeted to the qutE locus, is directly proportional to its copy number. The analysis of transformed strains in which single copies of lacZ fusions have been targeted to the qutE locus has identified three constitutive positively acting sequence elements in the pgk gene. Firstly, sequence located between -161 and -120bp (possibly spanning -120) relative to the transcript start site is essential for expression, thus demonstrating that the putative core promoter, including potential TATA and CCAAT boxes, and a pyrimidine rich region, is not alone sufficient to enable expression. A comparison of this sequence to known promoter elements has revealed the presence of an octamer AAGCAAAT (-131 to -124), with a seven out of eight base pair match to the consensus octamer sequence ATGCAAAT characterized as being essential for the expression of several higher eukaryotic genes. Whilst the octamer containing sequence contributes almost a residual 40% of the maximum expression recorded, a second region encompassing codons 14 to 183 and including the two introns of pgk has been shown to account for over 30% of the total activity. However, the full effect of the internal sequence may be greater, since the large 3-galactosidase fusion proteins studied here have been shown to be unstable. Thirdly, an extensive region extending from 0.5 to over 3Kb upstream of the start site has been shown to be required for full expression, accounting for almost 30% of the total recorded activity. Furthermore, sequence located between -638 and -488bp, and including an eight bp consensus element TGAGGTGT common to the four cloned A.nidulans glycolytic genes, has been shown to modulate expression: increasing activity about 1.5-fold on gluconeogenic compared to glycolytic carbon sources. In a separate exercise, the native pgk gene has been replaced with disrupted sequence to generate a pgk mutant strain of A.nidulans. This replacement has been achieved in a diploid strain, from which both pgk+ and pgk-segregants have been isolated, and the analysis of these has allowed the pgk locus to be assigned to chromosome VIII. The pgk- mutant strain that has been isolated is deficient in 3-phosphoglycerate kinase activity, requires both acetate and glycerol for growth, conidiates poorly, and is poisoned by even moderate concentrations of hexoses.Thu, 19 Nov 2015 08:53:44 GMThttp://hdl.handle.net/2381/344762015-11-19T08:53:44ZThe molecular genetics of cell shape in Escherichia coli.http://hdl.handle.net/2381/34475
Title: The molecular genetics of cell shape in Escherichia coli.
Authors: Stoker, Neil Graham.
Abstract: Abstract not available.Thu, 19 Nov 2015 08:53:43 GMThttp://hdl.handle.net/2381/344752015-11-19T08:53:43ZPredictors of hallux valgus: A study of heritability.http://hdl.handle.net/2381/34474
Title: Predictors of hallux valgus: A study of heritability.
Authors: Spooner, S. K.
Abstract: Hallux valgus is a complex progressive foot deformity of uncertain aetiology. The disorder is characterised by a lateral deviation of the hallux at the first metatarsophalangeal joint; an angle > 15 is considered as clinical hallux valgus. A model that predicts first metatarsophalangeal joint angle and thus, hallux valgus is potentially very useful; enabling the clinician to identify individuals at risk of developing the disorder and to predict prognosis. The aim of this study is to develop such a model. The literature relating to hallux valgus identifies eight potential aetiological factors of hallux valgus. The scientific evidence presented in support of these suspected aetiological factors, and the theories of pathology of hallux valgus in association with these factors were critically evaluated by a review of the literature. Methods to evaluate the significance of these factors in hallux valgus were identified and appraised. These methods were applied to a large sample of genetically related individuals. The genetic and environmental influences affecting first metatarsophalangeal joint angle, pes planus, metatarsal formula, digital formula and first ray neutral position were explored through the statistical analysis of the data obtained from the sample. The results of analyses suggest that all of these variables are gender influenced, multifactorial traits. Further analysis of a subset of data generated a statistical model that relates the degree of hallux deviation at the first metatarsophalangeal joint (and thus, the degree of hallux valgus) to clinically measurable predictor variables. A further subset of data was applied to test the model. The model was found to accurately predict first metatarsophalangeal joint angle in 92% of cases. Application of the model allows die clinician to evaluate an individual's risk of developing hallux valgus enabling accurate prognosis. Recommendations for achieving improved prognosis and the implications for future research are proposed.Thu, 19 Nov 2015 08:53:43 GMThttp://hdl.handle.net/2381/344742015-11-19T08:53:43ZThe killer system of Kluyveromyces lactis: Molecular genetic analysis of killer plasmid K2 gene function.http://hdl.handle.net/2381/34472
Title: The killer system of Kluyveromyces lactis: Molecular genetic analysis of killer plasmid K2 gene function.
Authors: Schaffrath, Raffael.
Abstract: K. lactis killer strains carry two cytoplasmic, linear dsDNA plasmids, termed k1 and k2. DNA sequence analysis has revealed the presence of 14 plasmid-encoded ORFs. All genes are transcribed independently and all are preceded by putative promoter elements, the UCS motifs. Plasmid genes are generally not expressed when cloned with their own promoters into yeast nuclear vectors; similarly, cytoplasmic expression of genes carrying nuclear promoters does not occur when they are placed on k1 or k2. To overcome this compartmental incompatibility I have constructed suitable UCS-marker gene fusions for k2 gene disruption analysis. Disruption of the putative DNA (k2ORF2) and RNA (k2ORF6) polymerase structural genes by integration of the the K1TRP1 gene fused to the k1UCS2 element as selection marker, yielded ORF2 and ORF6 deletion plasmids. ORF20 and ORF60 plasmids were unable to displace parental k2 during Trp+ selective growth indicating both genes to be essential for plasmid functionality. The hybrids were reduced in copy numbers relative to k2 with ORF20 more drastically affected than ORF60 plasmids implying direct involvement of ORF2 in plasmid replication and an indirect maintenance function for the ORF6 gene product. Similarly, disruption of k2ORF5 via integration of a k2UCS5-ScLEU2 marker gene yielded ORF5 deletion plasmids, termed rk2. ORF5 appears to be an essential gene for plasmid integrity since rk2 was unable to displace native k2 during Leu+ selective growth. By shuffling ORF5 from k2 onto k1 I have shown this essential gene to be functionally interchangeable between both episomes. Once transferred onto k1, ORF5 was fully able to complement the ORF50 deletion on rk2 in trans. ORF5 is a protein encoding gene as shown by shuffling an in vitro epitope-tagged allele. Western blotting identified a protein (19.5kDa) corresponding to the expected size of the tagged Orf5p product. Expression levels indicate Orf5p to be an abundant k2 product implying structural rather than regulatory function. The gene shuffle has been used to investigate UCS function. By transplacing various ORF5 deletion constructs, and analysing trans-complementation, a 40 bp k2 fragment including the UCS5 motif has been identified as a cis-acting promoter essential for k2 gene function and transcriptional activation of a ScLEU2 reporter gene. Analyses revealed a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart: cytoplasmic, UCS5-driven expression of ScLEU2 was non-repressible by leucine and reduced up to eight fold compared to fully derepressed nuclear K1LEU2 mRNA levels. Thus, k2 and k1 appear to employ a balanced low level expression system. Finally, the k2ORF7 (putative RNApol subunit) and k2ORF5 products were detected by over-expression of epitope-tagged alleles in E. coli and baculovirus systems. Western analysis identified proteins with apparent molecular weights of 18 and 20 kDa, corresponding in size to the predicted products.Thu, 19 Nov 2015 08:53:42 GMThttp://hdl.handle.net/2381/344722015-11-19T08:53:42ZActin-associated proteins in the yeast Saccharomyces cerevisiae.http://hdl.handle.net/2381/34473
Title: Actin-associated proteins in the yeast Saccharomyces cerevisiae.
Authors: Shiels, Gary.
Abstract: The MYO1 gene encodes the type II myosin heavy chain in the yeast Saccharomyces cerevisiae. The 5' end of the MYO1 gene was deleted to produce a myol null mutant (myo1). The resulting mutation was not lethal and cells displayed a phenotype that was indistinguishable from cells bearing a disruption in the 3' end of the gene, ?myol mutants are defective in cytokinesis, resulting in long chains of cells. Although nuclear division proceeds, there is a defect in nuclear migration resulting in cells containing multiple nuclei. The myo1 mutant tends to lyse when grown in liquid medium and was found to be sensitive to changes in pH. The mutant also showed defects in vacuolar morphology, chitin distribution and apparent defects in bud site selection. An attempt was made to identify other myosin heavy chain proteins by purifying actomyosin complexes. A 200kDa protein was co-purified with the myosin heavy chain following two rounds of purification. Polyclonal antibodies were raised against the purified protein and used to screen a yeast expression library. A single clone was isolated and identified as the yeast FAS1 gene. FAS1 encodes the beta-subunit of the yeast fatty acid synthetase. The disruption of the FAS1 gene confirmed that fatty acid synthetase was a component of actomyosin preparations and offers the potential for purifying large amounts of myosin heavy chain from yeast.Thu, 19 Nov 2015 08:53:42 GMThttp://hdl.handle.net/2381/344732015-11-19T08:53:42ZThe mode of action of colicin E2 with regard to the structure of the Escherichia coli cell envelope.http://hdl.handle.net/2381/34470
Title: The mode of action of colicin E2 with regard to the structure of the Escherichia coli cell envelope.
Authors: Samson, A. C. R.
Abstract: The importance of cell membranes in maintaining the structural integrity of living organisms has been known for some time. A very complex and important role for membranes as the spatial and temporal organizers of the cell's numerous metabolic functions is presently being unfurled. Although overall membrane structure may appear to be similar amongst the vast range of living organisms, the basic framework is necessarily adorned with specialized coordinated systems relevant to each cell's functions. The mechanism of colicin E2 interaction with the cell surface of Escherichia coli is examined and discussed. Colicin action upon the bacterial cell's biochemical processes is both indirect and highly specific, and is mediated by some transmission system in the bacterial cell membrane. Fluorescein labelled colicin E2 and ferritin labelled anti-E2 Y globulin have been prepared and used in an attempt to locate the positions of colicin E2 molecules in or on the cell surface of sensitive bacteria. Generalized disturbance of cell envelope integrity subsequent to colicin action has been sought but not found. The highly specific nature of colicin action upon the membrane is supported. Treatments designed to interfere uith bacterial cell envelope integrity perturb the action of colicin E2. Hembrane fractionation procedures suitable for studying bacterial membrane structure are presently becoming available. An additional membrane fractionation procedure is presented, based upon the interaction of cadmium N-lauroyl sarcosinate with bacterial cell extracts. Sodium dodecyl sulphate acrylamide gel electrophoretic separation of E. coli envelope proteins from mutants refractory to the action of colicin E2 has shoun that these membrane-containing structures have an altered protein component compared to the non-refractory parental strain. Hypotheses explaining a certain type of colicin E2 refractivity are presented. Future studies involving the use of colicins and genetic analysis as effective tools in probing the complexities of the E. coli cell membrane structure are outlined.Thu, 19 Nov 2015 08:53:41 GMThttp://hdl.handle.net/2381/344702015-11-19T08:53:41ZDevelopment of genetic tests for the rapid identification of species in foods.http://hdl.handle.net/2381/34471
Title: Development of genetic tests for the rapid identification of species in foods.
Authors: Sawyer, Jason Paul.
Abstract: The aim of the project was the development of DNA based tests that could be used to identify and distinguish common commercial meat species. The emphasis of the work was on development of a method that would be successful for cooked and highly processed foods. The potential of the PCR (polymerase chain reaction) as the basis of such a test was examined. The DNA sequence chosen as the target for PCR amplification was mitochondrial DNA (mtDNA). DNA sequence from a region of the mtDNA called the control region was obtained for cattle, sheep, pig, goat and chicken by DNA sequencing and from the literature. Using this information, species-specific PCR primers pairs were developed that allowed all the above species to be identified and distinguished. Amplification products were specific, showing no cross-reaction with other species, and were of a unique size for each species. In addition, multiplex PCR, using mixtures of species-specific primer pairs, allowed identification of several species in one PCR reaction. Amplification was successfully achieved from DNA extracted from blood, raw meat and food samples including cooked and highly processed foods and products. To assess the consistency of the species-specific primers, amplification was carried out on numerous individuals and breeds of particular species; amplification was successful for all individuals tested. DNA sequencing of the control region of individual animals was also carried out to assess the level of polymorphism within domestic meat species. These experiments showed that DNA polymorphism was low in domestic species and suggested that a species-specific PCR test based on the mtDNA control region would be consistent. The complete sequence of sheep mtDNA control region was obtained. The sheep control region is characterised by the presence of an area of repeated DNA consisting of four seventy five base pair repeats. Alignment of the control region sequence with other domestic species revealed that the putative mitochondrial control element CSB 1 is well conserved in these species. However, the control elements CSB 2 and 3 are not well conserved even in the closely related species of sheep, goat and cattle.Thu, 19 Nov 2015 08:53:41 GMThttp://hdl.handle.net/2381/344712015-11-19T08:53:41ZA study of human collagen genes and the molecular genetics of osteogenesis imperfecta.http://hdl.handle.net/2381/34469
Title: A study of human collagen genes and the molecular genetics of osteogenesis imperfecta.
Authors: Rose, Nicola Jayne.
Abstract: The human collagens are a family of related proteins which all possess at least one triple helical domain characterised by a Gly-Xaa-Yaa amino acid repeat motif where Xaa and Yaa are frequently proline and hydroxyproline respectively. This repetitive structure is determined by the characteristic sequence of the genes encoding the component polypeptides. Mutations in the COL1A1 and COL1A2 genes, encoding the proalpha1(I) and proalpha2(I) chains of type I collagen have been reported. These mutations can alter the polypeptide chains of type I collagen and interfere with the nature or amount of mature collagen produced by the cell and have been implicated as the cause of the connective tissue disorder osteogenesis imperfecta (brittle bone disease). This disorder is heterogeneous in nature with phenotypes ranging from mild to lethal in the perinatal period. In this study, single strand conformation polymorphism analysis was coupled with DNA sequencing in order to identify mutations in type I collagen genes which could be responsible for the disorder phenotype in a number of patients. Three novel glycine by serine substitutions were identified, two of which were found to be shared by more than one unrelated individual - a phenomenon rarely observed for osteogenesis imperfecta mutations. The fourth mutation was the first glycine by glutamic acid substitution reported to be the cause of this disorder. All of these substitutions occurred within the triple helical region of the proalpha2(I) chain. These data add to the knowledge of the relationship between the position and nature of substituting amino acids and the resulting phenotypes. In addition, two novel but neutral sequence variants were identified. A novel source of collagen mRNAs based upon basal transcription levels was also investigated as was an approach for identifying novel collagenous sequences using degenerate oligonucleotide-primed polymerase chain reaction amplification of DNA.Thu, 19 Nov 2015 08:53:40 GMThttp://hdl.handle.net/2381/344692015-11-19T08:53:40ZRegulation of leading region genes on IncI1 plasmid ColIb-P9.http://hdl.handle.net/2381/34468
Title: Regulation of leading region genes on IncI1 plasmid ColIb-P9.
Authors: Roscoe, Richard Alan.
Abstract: The psiB and ssb genes of IncI1 plasmid ColIb-P9 reside in the leading region, which is the first portion of the plasmid to be transferred during conjugation. These genes are expressed in a transient burst in newly-formed transconjugants via a process known as zygotic induction, and are thought to promote establishment of the immigrant plasmid. One hypothesis for the regulation of zygotic induction is that the genes are activated as part of a conjugation-induced heat-shock response. Both ssb and psiB are damage-inducible and have putative sigma 32-dependent promoter sequences. Data are presented showing that zygotic induction occurs when sigma 32 levels are limited and that conjugation fails to induce the heat- shock response, as measured using a sigma 32-dependent reporter construct. A second hypothesis is that zygotic induction results from a transient loss of negative supercoiling during plasmid transfer. The finding that neither psiB nor ssb expression is strongly induced in vegetative cells treated with coumermycin is inconsistent with this hypothesis. A third possibility is that psiB and ssb are regulated by a plasmid-encoded trans-acting repressor which is absent from the recipient cell. This hypothesis was tested with an entry-exclusion mutant of ColIb, created by insertional inactivation of the eex locus. When ColIb was transferred to a recipient harbouring this mutant, zygotic induction was observed. This finding also demonstrates that zygotic induction is independent of vegetative replication, since the incoming and resident plasmids are incompatible. Previous work suggested that psiB and ssb are closely linked to promoters. However, data obtained using RP4- and ColIb-mobilisable constructs carrying different portions of the leading region suggest that at least 5 kb of upstream DNA is necessary for zygotic induction of both genes, thus indicating that they are induced as part of a leading region operon.Thu, 19 Nov 2015 08:53:40 GMThttp://hdl.handle.net/2381/344682015-11-19T08:53:40ZA study on the beta-globin gene cluster in man and the primates.http://hdl.handle.net/2381/34467
Title: A study on the beta-globin gene cluster in man and the primates.
Authors: Barrie, Paul Alexander.
Abstract: Human haemoglobins are coded for by two unlinked clusters of alpha and beta-globin genes. The human beta-globin cluster appears to have evolved by a series of tandem gene duplications and contains considerable stretches of intergenic DNA possibly involved in the developmental control of globin gene expression. In order to understand the evolution of this cluster, the arrangement of primate beta-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to the great apes and these compared to the published genomic map of the human beta-globin gene cluster. The arrangement of the entire epsilonupsilonupsilonbeta beta-globin gene cluster in the gorilla, chimpanzee and yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequences divergence of approximately 1x10-9 nucleotide substitutions per nucleotide site per year as compared with the currently accepted silent site rate of 5x10-9 nucleotide substitutions per nucleotide site per year. The owl monkey, a New world species, has a single upsilon-globin gene suggesting that the upsilon-globin gene duplication in man is ancient and occurred 20 to 40 million years ago---a surprising contrast to the very high degree of sequence homology existing between these two genes. The owl monkey appears to possess duplicated beta-globin genes suggesting that the deltabeta gene duplication is between 40 and 70 million years old. The beta-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 17,000 base-pairs) and contains single epsilon-, upsilon- and beta-globin genes. The upsilon- and beta-globin genes in this species are separated by a mosaic gene ([psi]beta') containing the 3' end of a beta-globin gene preceded by sequences related to the 5' end of the epsilon-globin gene as determined by hybridisation studies. The brown lemur beta-globin gene cluster has been cloned into a bacteriophage lambda vector as a set of overlapping DNA fragments covering the entire region and the 'psibeta' gene sub-cloned into a plasmid vector. Initial sequencing the 3' end of this gene has further indicated its beta-like nature.Thu, 19 Nov 2015 08:53:39 GMThttp://hdl.handle.net/2381/344672015-11-19T08:53:39ZAerobactin in human and animal disease.http://hdl.handle.net/2381/34466
Title: Aerobactin in human and animal disease.
Authors: Roberts, Mark
Abstract: Aerobactin in human and animal disease Mark Roberts The aerobactin iron uptake system is specified by a cluster of five genes, four for the synthesis of the siderophore aerobactin and the fifth for its cognate outer membrane protein receptor. The aerobactin genes have been cloned and subjected to molecular analysis. The role of the product of one of these genes (the 50,000 dalton protein) was in dispute and had been reported to be involved in both the transport and the biosynthesis of aerobactin. Complementation studies showed that the protein is intimately involved in aerobactin biosynthesis. Also, strains carrying plasmids with transposon insertions that abolished production of the 50 K protein were still able to transport aerobactin, showing that the outer membrane receptor is the only protein specified by the aerobactin system necessary for the uptake of ferri-aerobactin. E. coli strains isolated from domestic animals were screened for aerobactin and colicin V production by bioassay and by colony hybridization using DNA probes derived from the aerobactin biosynthesis and colicin V activity regions of ColV-K30. There was a high incidence of aerobactin production among septicaemic strains compared with a very low incidence among faecal strains. Colicin V production correlated with aerobactin production but the association was not absolute. Southern hybridization experiments indicated that the aerobactin genes of animal strains almost always reside on large (usually ColV) plasmids. Diarrheagenic strains of E. coli were also screened for aerobactin production. No enterotoxigenic strains produced aerobactin but strains belonging to the enteropathogenic, enteroinvasive and facultatively enteropathogenic E. coli groups contained members that produced aerobactin. The aerobactin biosynthesis probe hybridized to small plasmids of enteropathogenic E. coli strains in Southern blots. Antisera were raised in rabbits to both native and denatured aerobactin receptor protein (IutA). The antisera raised to denatured protein reacted with denatured protein in Western blots (WB) and immunoprecipitation (IP) but did not inhibit aerobactin or cloacin binding to IutA. The serum raised against the native protein inhibited cloacin binding but did not react in WB or IP. The cloned aerobactin genes restored the virulence in a murine peritonitis model of a wild type animal strain of E. coli cured of its CoIV plasmid. Prior growth in iron-restricted medium enhanced the virulence of the infecting strain. None of the anti-IutA sera passively protected mice from infection with a wild type iutA+ strain.Thu, 19 Nov 2015 08:53:39 GMThttp://hdl.handle.net/2381/344662015-11-19T08:53:39ZCaMV gene expression: The analysis of two CaMV promoters in yeast and higher plants.http://hdl.handle.net/2381/34465
Title: CaMV gene expression: The analysis of two CaMV promoters in yeast and higher plants.
Authors: Richardson, Jennifer H.
Abstract: The aim of this study was to assess the feasibility of using the budding yeast Saccharomyces cerevisiae as a system in which to analyse plant promoters. The promoters chosen for study were the 19S and 35S promoters of cauliflower mosaic virus (CaMV) which, like cellular plant promoters, are transcribed in the plant nucleus by host cell RNA polymerase II. A complete CaMV genome was introduced into yeast on a 2 micron plasmid-based vector and using Northern blot analysis, several CaMV-hybridising transcripts were detected. More precise information on the activity of the promoters was obtained by constructing gene fusions in which the 19S and 35S promoters were linked to the bacterial lacZ gene. Biochemical assays for &deg;-galactosidase showed that cells harbouring the 19S-lacZ gene expressed &deg;-galactosidase but those harbouring the 35S-lacZ gene did not. The insertion of a yeast transcription termination signal upstream of the 19S promoter did not abolish or diminish expression of the 19S-lacZ gene. &deg;-galactosidase was present at low levels in cells expressing 19S-lacZ, constituting less than 0.01% of total cell protein. The 5'ends of 19S-lacZ transcripts present in yeast were mapped by primer extension. The major RNA species initiated approximately 250bp upstream of the 19S-lacZ coding region, indicating the existence of a fortuitous promoter in this region of the CaMV DNA. Two less abundant RNA species initiated within the 19S-lacZ open reading frame at positions +9 and +25bp and may be produced from the genuine 19S promoter. There is evidence to suggest that one or both of these shorter transcripts is the functional mRNA for &deg;-galactosidase. All three classes of RNA were polyadenylated. Coupling of the 19S-lacZ gene to a yeast enhancer (the GAL UAS) produced a 5-fold increase in &deg;-galactosidase activity. At the transcriptional level, activation of the enhancer resulted in a massive increase in the level of the RNA initiating at -250bp but had a minor influence of the levels of the two RNA species initiating at +9 and +25. A series of deletion mutations within the 19S promoter was constructed using Ba131 nuclease. Analysis of these mutations in yeast revealed that sequences from -500 to -193bp and from -137 to -62bp were not required for 19S promoter function, but a deletion from -62 to -21bp (which removes the putative TATA box) severely reduced 19S-1acZ gene expression. Transgenic tobacco plants containing the 19S promoter deletions fused to a CAT gene were produced by A.tumefaciens-mediated gene transfer but the analysis of these plants was not completed.Thu, 19 Nov 2015 08:53:39 GMThttp://hdl.handle.net/2381/344652015-11-19T08:53:39ZEvasion of restriction systems by plasmid ColIb-P9 during bacterial conjugation.http://hdl.handle.net/2381/34463
Title: Evasion of restriction systems by plasmid ColIb-P9 during bacterial conjugation.
Authors: Read, Timothy Damian.
Abstract: Conjugative transmission of enterobacterial plasmid ColIb was found to be relatively resistant to type I and type II restriction systems specified by the recipient cell. One process contributing to evasion of restriction barriers during conjugation apparently involves transfer of multiple copies of the plasmid. A more specialized anti-restriction mechanism requires the product of the ColIb ard (alleviation of restriction of DNA) gene. The Ard phenotype was discovered from the ability of ColIb to alleviate EcoK (type I) restriction of unmodified infecting the plasmid-harbouring cell. Ard activity alleviated restriction of phage by representatives of all three families of type I R-M system, but not by type II or type in systems. ColIb had no effect on the modification activity of EcoK but this activity was impaired by multicopy recombinant plasmids supporting overexpression of ard. A 498 nucleotide sequence corresponding to ard was identified. The Ard protein (apparent mass of c22 KDa) was visualised following in vitro transcription and translation of ard+ recombinant plasmids. Gene fusion studies indicated that only the C-terminal two-thirds of the Ard protein is necessary for the restriction alleviation phenotype. A ColIbdrd ard mutant was constructed by a gene replacement strategy. Properties of the plasmid showed that Ard protects ColIb from EcoK restriction following conjugative transfer and that protection requires expression of the gene on the immigrant plasmid. The ard gene is located in the leading region, which is the portion of ColIb transferred first in conjugation, and is near to the psiB and ssb loci. The latter two genes are known to be zygotically induced following transfer. However, studies with lacZ reporter probes showed that ard was not subject to zygotic induction; the gene is apparently expressed at very low levels before, and after conjugative transfer. It is proposed that ard facilitates the productive transfer of ColIb between its different natural enterobacterial hosts.Thu, 19 Nov 2015 08:53:38 GMThttp://hdl.handle.net/2381/344632015-11-19T08:53:38ZMolecular analysis of the mosquito larvicidal toxins of Bacillus sphaericus 1593M.http://hdl.handle.net/2381/34462
Title: Molecular analysis of the mosquito larvicidal toxins of Bacillus sphaericus 1593M.
Authors: Rajan, Vidya.
Abstract: Bacillus sphaericus produces a sporulation associated toxin specific for the larvae of dipteran insects: mosquitoes that are vectors for the transmission of human diseases such as malaria and filariasis. Two dissimilar DNA sequences conferring larvicidal activity on recombinant Escherichia coli had previously been cloned from B. sphaericus 1593M (Souza at al., (1988), J. Biotechnol., 7, 71-82). Proteins encoded by one of these DNA sequences are studied in this thesis. The DNA sequence cloned from B. sphaericus 1593M is here shown to encode two proteins of 41 and 59KDa that are together required for toxicity to mosquito larvae. These proteins were previously hypothesized to exist in the crystal of B. sphaericus as high molecular weight oligomers (Baumann et al., (1985), J. Bacteriol., 163, 738-747). I now show that these two proteins are distinct from the high molecular weight protein which appears to constitute the majority species of the crystal. I have identified this protein as the Surface layer protein of B. sphaericus. I also show that the 59KDa species is a distinct and separate constituent of the crystal. As a prerequisite for raising antibodies to study regulation of synthesis of these proteins, purification following overproduction or secretion was investigated. Thus, attempts were made to purify the 41 and 59KDa proteins from E. coli cells using the C-terminal signal sequence of Haemolysin, a protein normally secreted from E. coli. Furthermore, attempts were made to express the gene encoding the 4lKDa protein in E. coli to a high level from PR and PL promoters. I show that this protein could not be overexpressed in this way. This was attributed to a lack of transcript termination on the vector due to the presence of a faulty fd transcription terminator downstream of the gene. The nucleotide sequence of the gene encoding the 59KDa protein was determined. This sequence was then used to design an approach which led to the overexpression of the gene from the T7 RNA polymerase-recognized 10 promoter on the plasmid pET3a. This in turn allowed the production of antibodies. The expression of the mRNA transcripts of the genes encoding the 41 and 59KDa proteins were also studied in B. sphaericus and B-galactosidase fusions were constructed to serve as "reporters" for the expression of the 41 and 59KDa proteins in E. coli and B. subtilis. I propose that the regulation of expression of the genes is complicated, and depends upon the use of two, temporally regulated promoters.Thu, 19 Nov 2015 08:53:38 GMThttp://hdl.handle.net/2381/344622015-11-19T08:53:38ZA genetic analysis of the transfer genes of the inci1 plasmid colib-p9.http://hdl.handle.net/2381/34464
Title: A genetic analysis of the transfer genes of the inci1 plasmid colib-p9.
Authors: Rees, Catherine E. D.
Abstract: Plasmid ColIb-P9 is a 93.2 kb self-transmissible plasmid, belonging to the I1 incompatibility group. Whilst much data had been gained concerning the molecular biology of conjugation mediated by this plasmid, a lack of information exsisted concerning the genetic organisation of the transfer genes. A physical map of the plasmid was constructed by detailed restriction analysis of DNA fragments sub-cloned from ColIb-P9. These fragments were also used to locate the positions of the transfer gene sog and the origin of transfer. Transposons Tn5 and Tnl723 were used to construct insertion mutants at defined points in ColIb-P9 and the effect of these on the expression of various transfer-related functions was studied. Using this technique, the probable location of the genes encoding the thick and thin sex pili were identified and also the site of the plasmid-encoded nuclease gene. The exact location of the entry exclusion gene was also determined. Complementation studies using the sub-cloned fragments of ColIb-P9 and a set of cosmid-clones generated from ColIbdrd-1 indicated that a positive regulator of the expression of the transfer genes exsisted and that this was composed of two genetically distinct elements. Studies involving wild type ColIb-P9 (drd+) indicated that this positive regulatory system is subject to negative control in cells containing the drd+ plasmid. The information gained from these studies was combined into a model of the organisation of the transfer genes of ColIb-P9. This defines at least three separate Tra regions, covering some 50 kb of the plasmid, with the origin of transfer located at one end of the transfer region.Thu, 19 Nov 2015 08:53:38 GMThttp://hdl.handle.net/2381/344642015-11-19T08:53:38ZAnalysis of the structure and expression of mammalian myoglobin genes.http://hdl.handle.net/2381/34461
Title: Analysis of the structure and expression of mammalian myoglobin genes.
Authors: Price, Melanie.
Abstract: The myoglobin gene is a distant member of the globin gene superfamily, expressed in muscle rather than erythroid cells. In vertebrate muscle, myoglobin is the principle haemoprotein and serves to facilitate the diffusion of oxygen from the vascular system to the muscle mitochondria. A single gene encodes myoglobin of both cardiac and skeletal muscle in the mouse. The characterisation of the mouse myoglobin gene established that it has the same three exon/two intron structure found in the ?- and ?-globin genes but differs from haemoglobin genes in having very long non-coding DNA regions. These features are shared by human and seal myoglobin genes. Sequence comparison of human, seal and mouse myoglobin genes revealed a highly conserved upstream domain, approximately 120 bp before the myoglobin gene CAP site and extending over 200 bp. Although the position before the CAP site suggests it may play some regulatory role, this sequence has no homology to conserved 5'-flanking sequences described for contractile protein genes. As expected from the early appearance of low levels of myoglobin mRNA in embryonic skeletal muscle, myoglobin gene expression is induced in both rat and mouse embryonic myoblasts during differentiation in vitro, concomitant with the contractile protein genes. Myoglobin-CAT gene fusions containing -1 kb of 5'-myoglobin DNA, including the conserved domain, are expressed on introduction into mouse myoblasts and furthermore, expression increases during myoblast differentiation over a transient expression period. This should provide a good in vitro system to study myoglobin gene expression at the molecular level during myogenesis.Thu, 19 Nov 2015 08:53:37 GMThttp://hdl.handle.net/2381/344612015-11-19T08:53:37ZInvestigation of the tonB gene product of Escherichia coli.http://hdl.handle.net/2381/34460
Title: Investigation of the tonB gene product of Escherichia coli.
Authors: Plastow, Graham Stuart.
Abstract: The active transport of all ferric chelates and vitamin B12, infection by bacteriophages T1 and ?80, and killing by a group of colicins are dependent upon the tonB gene product. The initial stage in these processes is an energy-independent binding of the 'substrate' to specific receptor proteins in the outer membrane. Subsequent stages of transport require an energized inner membrane and the tonB gene product. The aim of this study was to identify the tonB product in order to investigate some of its properties directly. The use of ?-tonB transducing phages carrying wild type and mutant tonB alleles to program protein synthesis in UV-irradiated cells enabled the tonB product to be identified; a 40 kilodalton protein was no longer synthesised from tonB deletion and insertion mutants. This protein was associated with the sarkosyl soluble, inner membrane fraction of irradiated cells, suggesting that the translocation of ferric chelates etc. from their initial binding sites, at the exterior of the cells, is dependent upon an inner membrane protein. In an attempt to identify the tonB protein in vivo the tonB region was recloned into a runaway replication plasmid vector. Although cloned proteins were identified, the tonB product was not detected using this system. Physical maps were prepared for the phages and plasmids used in this study, and compared with those described by other workers. The approximate position of the tonB gene was also located. The gene products coded by the tonB region were also identified by utilizing an in vitro coupled transcription translation system. Interestingly, the mutant tonB genes programmed the synthesis of truncated polypeptides in this system. Finally, these results and those of other workers are drawn together to present some models for the action of the tonB product.Thu, 19 Nov 2015 08:53:37 GMThttp://hdl.handle.net/2381/344602015-11-19T08:53:37ZA genetic study of sugar metabolism and transport in Aspergillus nidulans.http://hdl.handle.net/2381/34458
Title: A genetic study of sugar metabolism and transport in Aspergillus nidulans.
Authors: Payton, Mark Anthony.
Abstract: On the basis of biochemical studies pyruvate kinase has been identified as a key regulatory enzyme in a number of organisms, however, there is little genetic evidence to substantiate this. In Aspergillus nidulans pyruvate kinase activity is high following growth on glycolytic carbon sources and low following growth on gluconeogenic carbon sources suggesting the enzyme is heavily regulated. This seemed an excellent model system to study the role and regulation of a key enzyme of carbon metabolism and a study was therefore undertaken involving the isolation of mutants of A.nidulans lacking pyruvate kinase activity and of strains producing pyruvate kinase constitutively. Following mutagenesis and filtration enrichment a number of mutants were isolated which failed to grow on glycolytic carbon sources but grew normally on gluconeogenic carbon sources. Ten such mutants lacked pyruvate kinase activity and identify a single gene locus, pkiA (chromosome V), probably the structural gene for the enzyme. The pyruvate kinase deficient pkiA mutants, though able to grow on gluconeogenic carbon sources, are poisoned by the addition of a glycolytic carbon source such as glucose or fructose, probably due to accumulation of a toxic metabolic product. A number of fructose-insensitive revertants of the pkiA1 strain were selected in an attempt to isolate strains defective in sugar transport. Amongst these revertants, two were shown to have altered sugar uptake characteristics, demonstrating that the reversion technique described is capable of generating bona fide sugar uptake mutants in A.nidulans. One interesting observation made during this work was that the presence of agar in growth media can generate spurious results. This was observed first for a number of acetate non-utilising (acu) mutants whose growth on a number of carbon sources was impaired by agar. It was subsequently demonstrated that A.nidulans can use agar as sole carbon source by the pathway of C2 metabolism.Thu, 19 Nov 2015 08:53:36 GMThttp://hdl.handle.net/2381/344582015-11-19T08:53:36ZMolecular evolution of a repetitive region within a clock gene in Drosophila.http://hdl.handle.net/2381/34459
Title: Molecular evolution of a repetitive region within a clock gene in Drosophila.
Authors: Peixoto, Alexandre Afranio.
Abstract: Abstract not available.Thu, 19 Nov 2015 08:53:36 GMThttp://hdl.handle.net/2381/344592015-11-19T08:53:36Z